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At least 595 records · Page 33Linked to original sources
PubMed1998

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases.

Trimeresurus stejnegeri venom which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin.

Blood Coagulation↗
PubMed1998

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper (Trimeresurus stejnegeri) venom.

From the venom of Chinese green tree viper (Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-1, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatography (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mol. wt of 50000, 31000 and 32000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnefibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA and stejnobin, stejnefibrase-1, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially Bbeta-chain while stejnefibrase-1 and -3 cleaved concomitantly Aalpha and Bbeta-chains of fibrinogen. None of these proteases degraded the gamma-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-1. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases.

Animals↗
PubMed1981

Comparison of alternative chromogens for renal immunohistochemistry.

Renal immunomicroscopy using enzyme labeled reagents has been shown to be a reliable method for the identification of immunoglobulins and complement in the routine evaluation of glomerular disease. However, the potential carcinogenicity of benzidine derivatives used in the procedure represents a major disadvantage of the technique. With a series of 55 renal biopsy specimens evaluated by light microscopy, immunofluorescence, and electron microscopy from patients with a variety of renal diseases, a study was done comparing aminoethylcarbazole and the Hanker-Yates reagent (p-phenylenediamine and pyrocatechol), chromogens chemically unrelated to benzidine. Aminoethylcarbazole was not suitable for renal immunomicroscopy, since in cases of antiglomerular basement membrane disease the color reaction product was finely granular at high magnification. Specimens immunostained with the Hanker-Yates reagent yielded permanent water insoluble reaction products and immunomicroscopic patterns identical to the results observed with immunofluorescence in all cases. To our knowledge, no carcinogenic properties have been identified for p-phenylenediamine or pyrocatechol. The Hanker-Yates reagent may be used routinely for renal enzyme immunomicroscopic studies with no currently identified carcinogenic hazard to laboratory personnel.

3,3'-Diaminobenzidine↗
PubMed2000

Asp41-His48 region of streptokinase is important in binding to a substrate plasminogen.

Streptokinase is a plasminogen activator protein produced by several strains of beta-hemolytic streptococci. Random mutagenesis of streptokinase was carried out for the determination of critical amino acid residues in plasminogen activation. We selected and sequenced 14 streptokinase mutants with no plasminogen activation activity on skim milk-plasminogen overlay plate. Specific activities of the selected streptokinase mutants were determined with chromogenic assay. Eight mutants (V19F, V35E, E85D, L292R, D325P, D341E, I345N, and M369L) resulted in greatly decreased amidolytic activities. However, unexpectedly, six mutants (D41C, S44K, S44P, R45P, H48T, and D220G) showed substantial amidolytic activities comparable to that of wild type. Moreover, five-point mutations were concentrated on the Asp41-His48 region. These data indicate that the Asp41-His48 region in a streptokinase-plasminogen binary complex plays an important role in binding to a substrate plasminogen.

Amidohydrolases↗
PubMed2000

Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate.

Sulfation of curdlan, a natural, linear beta-1,3-glucan results in potent anticoagulant and antithrombotic agents. The different activity characteristics in the classical coagulation assays were shown to depend on various structural parameters. To obtain more detailed information about their structure-dependent mechanisms of action, one representative of these beta-1,3-glucan sulfates (CurS), was further investigated using several coagulation assays and amidolytic tests with chromogenic substrates. The mode of action of CurS differs from that of heparin. CurS reduces the thrombin formation by principally inhibiting the intrinsic FXa generation. As shown by amidolytic assays, it eliminates already generated thrombin mainly by accelerating the HCII-mediated thrombin inactivation, whereas its AT-mediated anti-thrombin activity is considerably lower than that of heparin. Further, it prevents the thrombin-mediated fibrin polymerization by directly interfering with the thrombin action on fibrinogen as well as by binding to fibrinogen. Finally, CurS is capable to activate the contact system with the consequence of a potential fibrinolytic effect. In conclusion, beta-1,3-glucan sulfates do not inhibit the blood coagulation nonspecifically due to their anionic character, but in dependence on their individual structure, they interfere specifically with the coagulation process at several sites.

Amidohydrolases↗
PubMed2000

Effects of a fucoidan on the activation of plasminogen by u-PA and t-PA.

The effect of an anticoagulant fucoidan (C-I-H) from the brown seaweed Ecklonia kurome on the fibrinolytic system was studied in vitro using S-2251 as a substrate of plasmin. C-I-H enhanced the activation of Glu- and Lys-plasminogen by high molecular weight urokinase-type plasminogen activator (HMW u-PA) very effectively, but the activation by low molecular weight u-PA was hardly enhanced with C-I-H. C-I-H also potentiated moderately the activation by single- and two-chain tissue-type plasminogen activators (sct- and tct-PA). These effects of C-I-H were higher than those of heparin used. But C-I-H had no effect on the amidolytic activity of plasmin to S-2251. These results indicate that C-I-H promotes the generation of plasmin in the plasminogen activation by HMW u-PA and t-PA, but not the activity of generated plasmin. Kinetic analyses suggest that C-I-H enhances the HMW u-PA-mediated plasminogen activation by increasing the affinity of the activator for Glu- and Lys-plasminogen and by increasing the molecular activity of the activator. On the other hand, C-I-H had no effect on the affinity of tct-PA for both plasminogens. The catalytic efficiencies of HMW u-PA and tct-PA for the activation of both plasminogens were increased with C-I-H about 8- and 2-fold, respectively. The present results suggest that C-I-H has the fibrinolytic activity by stimulating the plasminogen activation by HMW u-PA and t-PA. The mechanism of the enhancement effect of C-I-H on the activation is presumed to be that C-I-H binds to plasminogen, thereby inducing a structural change of plasminogen susceptible to the action of plasminogen activators.

Anticoagulants↗
PubMed2000

Hirudin determination in plasma can be strongly influenced by the prothrombin level.

Recombinant hirudin is increasingly used for therapeutic and prophylactic anticoagulation. Several laboratory methods are available to measure r-hirudin, including clot-based and amidolytic methods. The snake venom ecarin converts prothrombin to meizothrombin. Hirudin inhibits meizothrombin, causing a prolongation of the ecarin clotting time (ECT). Because the ECT depends on prothrombin levels in plasma, it was compared with a chromogenic substrate assay (CSA) for the determination of r-hirudin levels in prothrombin deficient plasma samples. R-hirudin (0.0-2.0 microg/mL) was added to plasma samples with decreasing prothrombin concentrations (100-0%). Using the ECT, false high r-hirudin levels were observed even in r-hirudin-free plasma, when prothrombin levels were below 50%. This effect was more pronounced with increasing r-hirudin levels. Additionally, r-hirudin (0.5 microg/mL) was added to plasma of patients with acquired prothrombin deficiency due to oral anticoagulation. Hirudin levels were also overestimated in these plasma samples using ECT. In plasma samples of patients treated with r-hirudin, because of suspected heparin-induced thrombocytopenia (HIT), hirudin levels were already measured falsely high, when the prothrombin levels were below 70%. The chromogenic substrate assay (CSA) determined correct values in all prothrombin-deficient plasma samples. Therefore, the CSA should be used for hirudin level determination, if overestimation due to prothrombin deficiency should be avoided.

Antithrombins↗
PubMed2001

Evaluation of a sensitive colorimetric FXIII incorporation assay. Effects of FXIII Val34Leu, plasma fibrinogen concentration and congenital FXIII deficiency.

There is an increasing interest in the role of coagulation factor XIII (FXIII) in cardio- and cerebrovascular diseases. It has recently been reported that a common G-->T point mutation in the A-subunit gene of FXIII, which codes for a valine (val) to leucine (leu) change (FXIIIVal34Leu), is protective against thrombotic diseases but seems to increase the risk of intracerebral bleeding. We developed a colorimetric incorporation assay for detection of FXIII activity based on incorporation of 5-(biotinamido) pentylamine (BAPA) into fibrin or fibrinogen. With this new assay, we studied the effects of FXIIIVal34Leu mutation, plasma fibrinogen concentration and congenital FXIII deficiency on FXIII activity. There are no data available about the ability of different FXIII assays to detect altered activity in FXIIIVal34Leu genotypes. We therefore compared our results determined by the incorporation method with a commonly used photometric method based on ammonia release after cross-linking of glycine-ethylester to a specific glutamine containing peptide substrate. We also determined FXIII A-subunit antigen (Ag) levels using enzyme-linked immunosorbent assay (ELISA) technique. The FXIIIVal34Leu genotype could not be detected either by the photometric method nor by the FXIII A-subunit ELISA. The incorporation assay showed an increased specific FXIII activity in subjects possessing the leu allele. The photometric assay and ELISA gave similar results independent from genotype. In patients with congenital FXIII deficiency before and after substitution, however, ELISA and the incorporation assay gave similar results, whereas the photometric assay showed consistently higher values. Our results show that the incorporation assay, not the photometric assay based on ammonia release, can be used for detection of elevated activity in subjects with FXIIIVal34Leu. Because of specificity and over a wide range sensitivity, the assay can also be used for determination of FXIII deficiency and monitoring of FXIII substitution therapy.

Amines↗
PubMed2001

Avidin-biotin ELISA for measurement of prothrombin in human plasma.

In this study, IgG was partially purified from rabbit antisera against human normal prothrombin. The presence of antibodies against prothrombin was shown by Ouchterlony's double immunodiffusion method. This IgG was adsorbed to microwells and an enzyme-linked immunosorbent assay (ELISA) method was developed by using biotinylated prothrombin. We used alkaline phosphatase as the labeling enzyme. Plasma prothrombin concentrations in 30 healthy individuals and 15 patients with liver cirrhosis were measured using this ELISA method. Prothrombin levels of the cirrhotic patients (61+/-36 microg/ml) were significantly lower than those of the control values (117+/-43 microg/ml) (P<.001). In addition to the ELISA method in the same samples, we assayed prothrombin levels by nephelometry and prothrombin activities using staphylocoagulase and chromogenic substrate. With nephelometry, in healthy individuals, a mean value of 104+/-17 microg/ml and, in cirrhotic patients, a mean value of 51+/-26 microg/ml of prothrombin levels were detected. The prothrombin activities were 10532+/-2429 and 2433+/-330 U/l, respectively. In both methods, the prothrombin values of cirrhotic patients were also significantly lower than those of the healthy individuals (P<.001). The correlation coefficient between ELISA and nephelometry was r=.90, P<.01, and between ELISA and the amidolytic assay was r=.69, P<.01. Prothrombin times (PT) of cirrhotic patients (18.5+/-2.3 s) were significantly longer and albumin (2.4+/-0.5 g/dl) levels were significantly lower than those of normal values (P<.001). PT were negatively correlated with prothrombin levels assayed by ELISA (r=-.59, P<.01). This enzyme immunoassay technique can be used to measure prothrombin levels in plasma.

Animals↗
PubMed2001

Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modulated activation of protein C in vitro.

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human alpha-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl(2). TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K(D)) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC(50) values+/-S.D. of 4.4+/-0.8, 20.0+/-1.1 and 6.4+/-0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC(50) value of 290+/-10 nmol/l, which was enhanced fourfold (IC(50) 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.

Antithrombins↗
PubMed2002

Thrombin-activatable fibrinolysis inhibitor antigen and TAFI activity in patients with APC resistance caused by factor V Leiden mutation.

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as procarboxypeptidase U or plasma procarboxypeptidase B, is a relatively recently described plasma glycoprotein synthesised in the liver. It can be activated into active enzyme TAFIa (carboxypeptidase U or plasma carboxypeptidase B) by a complex of thrombin/thrombomodulin. TAFIa can potentially inhibit fibrinolysis by removing carboxyterminal lysine residues from partially degraded fibrin, decreasing plasminogen binding on the surface of fibrin, which thereby results in a decrease of the fibrinolytic activity. Since TAFI represents a connection between coagulation and fibrinolysis, it can be expected that TAFI levels are altered in different thrombotic and hemorrhagic diseases. Thrombin generation is increased in patients with activated protein C (APC) resistance, while it has been shown that APC has profibrinolytic effect. Therefore, changes in TAFI level should be found in patients with APC resistance due to factor V Leiden (FV Leiden) mutation. TAFI antigen (including TAFI, TAFIa and the inactive form TAFIai) and TAFI activity were determined in 17 female patients heterozygous for FV Leiden mutation while 13 healthy volunteers were controls. No statistically significant difference in levels of TAFI antigen was observed. TAFI activity was significantly reduced in APC resistance patients compared to control (P=.018). The nondifference in TAFI antigen, together with the decrease of TAFI activity level, can be explained by activation of TAFI to TAFIa and shifting of equilibrium towards an increase of the latter. This can be an indirect proof that TAFIa is increased in patients with APC resistance due to FV Leiden mutation, indicating that downregulation of fibrinolysis can be an additional risk factor for thrombosis in these patients.

Activated Protein C Resistance↗
PubMed2002

Separation of active and inactive forms of human antithrombin by heparin affinity chromatography.

During the manufacturing of an antithrombin preparation, it is necessary to define all steps that may damage or alter the target molecule, and thus decrease the biological activity of the inhibitor in blood coagulation. Pasteurization, commonly used procedure for viral inactivation of plasma derived antithrombin concentrates, was shown to partially alter the conformation of the active native antithrombin to an inactive latent form. To study intensively the different forms of inactive antithrombin that are formed upon heat treatment, human alpha-antithrombin, human beta-antithrombin and an equimolar mixture of the two isoforms were incubated at 60 degrees C for 15 h in the presence of citrate as stabilizing agent. Using two subsequent heparin affinity chromatography steps, three different inactive fractions were separated. By comparison of the heparin binding capacities, isoelectric points and unfolding characteristics of these inactive forms, the alpha-latent and beta-latent antithrombin isoforms could be identified. It was also shown that additional inactive forms such as proteinase cleaved and/or oxidized forms of antithrombin are formed during the heat treatment process. In four commercially available antithrombin preparations, all produced by pasteurization, the amount of inactive protein varied between 0.5% and 9.5%.

Antithrombin III↗
PubMed2002

Anti-Xa activity in obese patients after double standard dose of nadroparin for prophylaxis.

We measured anti-Xa activity before and at 2, 4, 9 and 12 h after subcutaneous injection of 0.3 ml nadroparin (2850 IU anti-Xa) in normal-weight volunteers (median bodyweight 71 kg, n=5) and compared them to the anti-Xa activity after subcutaneous injection of 0.6 ml nadroparin (5700 IU anti-Xa) in obese patients (median bodyweight 134 kg, n=8). The resulting median anti-Xa activity after 4 h was 1.4 times greater in the group of obese patients. This finding suggests that a linear, weight-adjusted increase in the prophylactic dose of nadroparin does not result in a linear increase of anti-Xa activity in obese patients.

Anticoagulants↗
PubMed1996

Regulation of single chain urokinase by small peptides.

Whether single chain urokinase (scuPA) expresses intrinsic enzymatic activity continues to be a subject of controversy. We report that the activity of scuPA is enhanced by a small plasmin substrate, H-D-valyl-L-leucyl-L-lysine-p-nitroanilide diacetate (D-VLK-p), but not by a second plasmin substrate, H-D-norleucyl-hexahydrotyrosyl-lysine-p-nitroanilide diacetate (*L*YK-p). D-VLK-p had no effect on the activity of a plasmin insensitive scuPA variant (scuPA-glu158) indicating that native scuPA can be cleaved by plasmin even at saturating concentrations of D-VLK-P. In contrast, D-VLK-P inhibited the activity of the native and scuPA-glu158 complexed with soluble urokinase receptor. Further, D-VLK-p stimulated the enzymatic activity of low molecular weight scuPA (amino acids 144-410) suggesting that D-VLK-P interacts with a second, previously undescribed regulatory site in scuPA.

Chromogenic Compounds↗
PubMed1997

Amyloid beta peptides stimulate tissue-type plasminogen activator but not recombinant prourokinase.

Tissue-type plasminogen activator (rt-PA) and prourokinase (rscu-PA) have been tested with respect to the influence of amyloid beta peptides on plasminogen activation which was monitored by cleavage of the chromogenic plasmin substrate S-2251. It was shown that rt-PA is stimulated by amyloid beta peptides at concentrations of 10 micrograms/ml in contrast to prourokinase, which does not alter its catalytic properties in presence of amyloid beta peptides. The stimulation of rt-PA can be inhibited by tranexamic acid indicating a molecular mode of stimulation similar to the fibrin mediated stimulation of rt-PA.

Amyloid beta-Peptides↗
PubMed1997

Partially purified Echis carinatus venom cleaves active-site-mutated bovine prothrombin at two sites.

Partially purified snake venom of Echis carinatus (ECV) is a well-known enzyme used to generate meizothrombin from prothrombin. In this reaction, meizothrombin is slowly converted to two smaller fragments. It is not certain if this slow reaction is catalyzed by meizothrombin or by ECV. Using previously constructed active site mutant bovine prothrombin, we eliminated meizothrombin autolysis and examined ECV reactions. ECV did slowly generate two fragments during activation of the active site mutant prothrombin. This result indicates that ECV cleaved bovine prothrombin at two sites: at Arg323-Ile to expose the active site, at another bond at or near the known meizothrombin autolysis site. Because the former cleavage is faster than the latter one, ECV is still a reasonable tool to generate meizothrombin under proper conditions.

Animals↗